Drosha interacts with a dsRNA-binding protein encoded by DiGeorge syndrome critical region 8 (DGCR8) to form the Microprocessor complex, which initiates miRNA biogenesis by chopping hairpin-shaped miRNA precursors (pre-miRNA) off the primary transcripts (pri-miRNA) in the nucleus. Regulation of miRNA function happens at the first step of its biogenesis. Reduced enzymatic activity of Drosha has been described in various malignancies. We created a set of reporters bearing various pri-miRNAs in the 3'UTR to measure the cleavage activity of microprocessor in vivo. Applying these reporters, we plan to identify novel cellular regulators of Drosha activity via genome-wide genetic screens. Using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), we generated a set of knockout cells in which various miRNA biogenesis genes, including Drosha and DGCR8, are inactivated. This enables us to monitor the cleavage events in cells without the interference from endogenous Drosha. We plan to construct a set of plasmids expressing Drosha, Dicer and DGCR8 mutants identified in cancer patients. Their impact on the efficiency and accuracy of miRNA biogenesis will then be tested by genetic complementation assays performed in the corresponding KO cells. To elucidate the molecular mechanisms, we have set up collaboration with Dr. Xinhua Ji (CCR/NCI) to determine crystal structures of human microprocessor in complex with pri-miRNAs substrates. Based mainly on non-cellular experiments, the current model posits that Drosha's activity is restrained inside the nucleus. By studying Drosha cleavage in living cells, we gather evidence to establish the existence of cytoplasmic Drosha (c-Drosha) activity. More importantly, we reveal alternative splicing as an underlying mechanism to induce c-Drosha activity. We identify a novel Drosha isoform in which the exon containing the putative nuclear localization signal (NLS) is skipped. This isoform is abundant in multiple cell lines and its levels vary dramatically among different human tissues, suggesting that c-Drosha has unique biological functions in gene regulation. Further analysis indicates that c-Drosha expression is upregulated in multiple cancer tissues, raising an intriguing possibility that c-Drosha functions as an oncogene by targeting mRNAs of tumor-suppressor genes for degradation. We will collaborate with Dr. Markus Hafner (NIAMS) to identify the direct targets of c-Drosha and Dr. Peng Yu (UT Austin) to further investigate c-Drosha's role in cancer development. DNA-directed RNAi (shRNA expressed from plasmids) is more desirable than traditional synthetic siRNAs in certain settings such as genetic screen and gene therapy. However, unsatisfactory knockdown efficacy and off-target effects hamper its applications, often due to inefficient, low fidelity of processing. Our previous finding on Dicer processing has established a loop-counting rule, which laid the groundwork in designing Pol III-driven pre-miRNA-like shRNAs free of the off-target effects resulting from heterogeneous processing. We are currently working on transferring such a design into the Pol II system, in which more complicated manipulation of shRNA function is possible. In particular, we are developing conditionally activated shRNAs whose function can be specifically turned on in cancer cells. This approach will dramatically increase shRNA specificity and safety. Given the long half-lives of mature miRNAs (ranging from hours to days), biogenesis control by itself is inadequate in situations that require rapid changes in miRNA function. Post-maturation regulation is an important component of how miRNAs function. Thus, in alignment with our goal of understanding miRNA regulation, we study the biogenesis and function of 3' isomiRs, which are miRNA variants generated by post-maturation tailing (adding nucleotides) and/or trimming (removing nucleotides). Based on our expertise of Drosha/Dicer processing, we have developed an approach to map Drosha cleavage sites on pri-miRNAs. Information gained is used as a reference to study post-maturation modifications on miRNAs, making it possible to accurately annotate 3' isomiRs in cancer. To investigate the function of 3' isomiR, we checked the status of 3' sequence modifications during miRNA overexpression and decay. We found that isomiR profiles are not random but rather tightly regulated. Interestingly, when the level of overexpressed miR-23a peaked around 60hr post-transfection, its isomiR profile became similar to endogenous miR-23a, despite the fact that level of the former was over a hundredfold more than the latter. This suggests that the 3' end modification is not merely a consequence of miRNA overexpression, but rather plays an active role in maintaining the miRNA hemostasis. To establish a causative relation, we are working on identifying the enzymes that are responsible for producing certain isomeric forms. We also aim to develop novel strategies to monitor the function specific to certain isomiRs. Overall, these studies seek to significantly advance our basic understanding of isomiRs, and provide a foundation for future mechanistic study of their functions in cancer.